In contrast to the extensive studies of CD4+ cells, mechanisms by which tolerance of mature CD8+ cells might be induced have been relatively little explored. This project will focus on characterizing two potential mechanisms for inducing peripheral non-responsiveness in CD8+ cytolytic T lymphocytes, antigen-activated cell death and anergy. Studies described in Aim 1 will employ artificial cell surface constructs to determine the antigen and ligand requirements for induction of activation-driven cell death by apoptosis and for anergy induction. Based on preliminary results, experiments will also be done to test the hypothesis that the same stimuli which activate positive responses at low or moderate levels induce T cell death (deletion) at high levels; i.e. when too 'strong' a stimulus is provided. Experiments will also be done to determine if CD8+ cells capable of producing IL-2 can be converted to an anergic state resembling that which occurs for TH1 cells, and will define the qualitative antigen and ligand requirements for induction of this state. Under Aim 2, use of artificial cell surface constructs bearing defined ligands will be used to determine the requirements for in vivo induction of non-responsiveness in class 1 restricted CD8+ cells. Previous work has shown that administration of microspheres bearing affinity-purified class 1 antigen and other cell surface ligands can significantly augment in vivo CTL response levels, and the properties of this strongly suggest that it results from precursor CTL recognition of the antigen on the microspheres. Preliminary results have been obtained in a peptide priming system which strongly suggest that CD8+ non-responsiveness is induced when the antigen-bearing microspheres provide too 'strong' a stimulus. This will be further examined, guided by the results obtained under Aim 1. These proposed studies have the potential to provide insight into the mechanisms by which mature CD8+ cells are rendered non-responsive to antigen, and suggest approaches for either inducing tolerance in transplantation or autoimmunity, or avoiding tolerance in peptide-antigen approaches to vaccination and immunotherapy.